Constructs of flipped SNAREs. The pre-prolactin signal sequence (SS) was fused to the N-termini of VAMP3, syntaxin4 and SNAP-23. A Myc tag was inserted between the signal sequence and the N-termini of VAMP3 and syntaxin4. A FLAG tag was engineered between the N-terminus of SNAP-23 and the signal sequence. In flipped syntaxin4(Δ111), the first 111 amino acids of syntaxin4 were deleted. In flipped syntaxin4 H3, the first 192 amino acids of syntaxin4 were deleted. In flipped SNAP-23, the three putative N-glycosylation sites (Asn3, Asn88, and Asn154) were eliminated by point mutations to Ala. The five cysteines in SNAP-23 were replaced with serines (C→S). TMD stands for transmembrane domain. The coiled-coil segments represent the SNARE motifs. Flipped syntaxin1 H3 and flipped SNAP-25 [10] were included for comparison.